Methods and devices for analyte collection, extraction, concentration, and detection for clinical applications

ABSTRACT

In various embodiments devices and methods for the detection and/or quantification of clinically relevant pathogens (e.g., bacteria, fungi, viruses, etc.) are provided. In certain embodiments the device comprises a lateral-flow assay that detects the bacterium at a concentration of less than about 6×106 cells/mL, less than about 3×106 cells/ml, less than about 1×106 CFU/mL, or less than about 50 μg/mL. In certain embodiments the device comprises an aqueous two-phase system (ATPS) comprising a mixed phase solution that separates into a first phase solution and a second phase solution; and a lateral-flow assay (LFA). In certain embodiments the device comprises a flow-through system comprising a concentration component comprising an aqueous two-phase system (ATPS) comprising a mixed phase solution that separates into a first phase solution and a second phase solution; and a detection component disposed beneath said concentration component.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/756,542, filed onFeb. 28, 2018, now U.S. Pat. No. 11,287,426 issued on Mar. 29, 2022which is a U.S. 371 National Phase of PCT/US2016/050257, filed on Sep.2, 2016, which claims benefit of and priority to U.S. Ser. No.62/214,801, filed on Sep. 4, 2015, each of which is incorporated hereinby reference in its entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[Not Applicable]

BACKGROUND

Assays have been used to detect the presence or the concentration ofvarious substances or pathogens in biological fluids. In a solid phaseimmunoassay, a receptor, typically an antibody which is specific for theligand to be detected, is immobilized on a solid support. A test fluidthat may comprise the analyte to be detected is contacted with the solidsupport and a receptor-analyte pair is formed when the target analyte ispresent. In order to make the receptor-ligand pair visible, labeledantibodies may be used that bind to the receptor-ligand pair followed byvisual detection of the labeled antibody bound to the receptor-ligandpair.

In so-called sandwich immunoassays, the analyte is typically sandwichedbetween a labeled antibody and an antibody immobilized on a solidsupport.

Porous materials such as nitrocellulose, nylon, cellulose acetate, glassfibers and other porous polymers have been employed as solid supports insolid phase immunoassays. In so-called lateral-flow assays, a fluidwherein the analyte is to be detected is applied to one end of a porousmembrane layer and flows in lateral direction through the membrane underthe action of capillary forces to be captured by an immobilized“receptor” that is capable of binding the analyte to be detected.

A general issue with lateral-flow immunoassays is assay sensitivity andtherewith signal intensity.

SUMMARY

In various embodiments devices and methods for the detection and/orquantification of clinically relevant pathogens (e.g., bacteria, fungi,viruses, etc.) are provided. In certain embodiments the device comprisesa lateral-flow assay that detects the bacterium at a concentration ofless than about 6×10⁶ cells/mL, less than about 3×10⁶ cells/ml, lessthan about 1×10⁶ CFU/mL, or less than about 50 μg/mL. In certainembodiments the device comprise an aqueous two-phase system (ATPS)comprising a mixed phase solution that separates into a first phasesolution and a second phase solution; and a lateral-flow assay (LFA). Incertain embodiments the device comprises a flow-through systemcomprising: a concentration component comprising an aqueous two-phasesystem (ATPS) comprising a mixed phase solution that separates into afirst phase solution and a second phase solution; and a detectioncomponent disposed beneath said concentration component.

Various embodiments contemplated herein may include, but need not belimited to, one or more of the following:

Embodiment 1: A device for the detection and/or quantification of abacterium, a fungus, or a virus in a sample, the device comprising alateral-flow assay that detects the bacterium at a concentration of lessthan about 6×10⁶ cells/mL, less than about 3×10⁶ cells/ml, less thanabout 1×10⁶ CFU/mL, or less than about 50 μg/mL.

Embodiment 2: A device for the detection and/or quantification of abacterium, a fungus, or a virus in a sample, said device comprising anaqueous two-phase system (ATPS) comprising a mixed phase solution thatseparates into a first phase solution and a second phase solution; and alateral-flow assay (LFA).

Embodiment 3: The device of embodiment 2, wherein the LFA comprises aporous matrix that is configured to receive and/or contain an ATPS orcomponents thereof.

Embodiment 4: The device according to any one of embodiments 2-3,wherein said LFA comprises a conjugate pad, a test line comprising anantibody that binds said bacterium, a control line comprising asecondary antibody, optionally an absorbent pad, and optionally a samplepad.

Embodiment 5: A device for the detection and/or quantification of abacterium, a fungus, or a virus in a sample, said device comprising: aflow-through system comprising:

-   -   a concentration component comprising an aqueous two-phase system        (ATPS) comprising a mixed phase solution that separates into a        first phase solution and a second phase solution; and    -   a detection component disposed beneath said concentration        component.

Embodiment 6: The device of embodiment 5, wherein said concentrationcomponent comprises one or more layers of a paper.

Embodiment 7: The device according to any one of embodiments 5-6,wherein said detection component comprises a conjugate pad, a reactionpad, and optionally a sink.

Embodiment 8: The device according to any one of embodiments 1-7,wherein said LFA or said flow-through system detects said bacterium inless than about 10 minutes.

Embodiment 9: The device according to any one of embodiments 1-8,wherein said device is configured for the detection of a bacterium.

Embodiment 10: The device of embodiment 9, wherein said bacterium is anoral bacterium, a bacterium found in urine, a bacterium found in vaginalfluid, or a bacterium found on a vaginal swab, or a bacterium found onan endocervical swab.

Embodiment 11: The device of embodiment 10, wherein said bacterium is anoral bacterium.

Embodiment 12: The device of embodiment 11, wherein said oral bacteriumcomprises Prevotella sp. (e.g., Pr. intermedia, Pr. nigrescens, etc.),Porphyromonas sp. (e.g., Porph. gingivalis, etc.), Streptococcus sp.(e.g., S. mutans, etc.), Actinomyces viscosus, Lactobacillus casei,Staphylococcus aureus, Coandica albicans, Lactobacillus acidophilus,Capnacytophaga gingivalis, Fusobacteriun nucleatum, or Bacteroidesfortsythus.

Embodiment 13: The device of embodiment 10, wherein said bacterium is abacterium found in vaginal fluid.

Embodiment 14: The device of embodiment 13, wherein said bacteriumcomprises Trichomonas sp., Actinomyces sp., Gardnerella sp., Neisseriasp., Chlamydia sp., or Treponema sp.

Embodiment 15: The device of embodiment 10, wherein said bacterium is abacterium found in urine.

Embodiment 16: The device of embodiment 15, wherein said bacteriumcomprises E. coli, Proteus sp., Trichomonas sp., Actinomyces sp.,Gardnerella sp., Neisseria sp., Chlamydia sp., or Treponema sp.

Embodiment 17: The device according to any one of embodiments 11-12,wherein the LFA or the detection component comprises an antibody thatdetects S. mutans.

Embodiment 18: The device according to any one of embodiments 1-17,wherein said ATPS is combined with said sample before application tosaid device.

Embodiment 19: The device according to any one of embodiments 1-17,wherein said ATPS is dehydrated on the lateral-flow assay or in theconcentration component of the flow-through assay before the device iscontacted with the sample.

Embodiment 20: The device according to any one of embodiments 1-19,wherein the ATPS comprises a mixed phase solution that separates into afirst phase solution and a second phase solution after the device iscontacted with the sample.

Embodiment 21: The device according to any one of embodiments 1-20,wherein the ATPS comprises a micellar/surfactant solution.

Embodiment 22: The device of embodiment 21, wherein the first phasesolution is concentrated in surfactant and the second phase solution hasa low concentration of surfactant.

Embodiment 23: The device according to any one of embodiments 1-20,wherein the first phase solution comprises a polymer and the secondphase solution comprises a surfactant.

Embodiment 24: The device of embodiment 23, wherein said polymercomprises dextran.

Embodiment 25: The device according to any one of embodiments 23-24,wherein the surfactant comprises a non-ionic surfactant or analkylpolyglycolether surfactant.

Embodiment 26: The device according to any one of embodiments 23-24,wherein the surfactant comprises a non-ionic surfactant nonionicsurfactant that has a hydrophilic polyethylene oxide chain and anaromatic hydrocarbon lipophilic or hydrophobic group (e.g., a Triton-Xsurfactant).

Embodiment 27: The device according to any one of embodiments 1-20,wherein the first phase solution comprises a first polymer and thesecond phase solution comprises a second polymer.

Embodiment 28: The device of embodiment 27, wherein the first/secondpolymer comprises polyethylene glycol, polypropylene glycol, or dextran.

Embodiment 29: The device according to any one of embodiments 1-20,wherein the first phase solution comprises a polymer and the secondphase solution comprises a salt.

Embodiment 30: The device of embodiment 29, wherein the first phasesolution comprises polyethylene glycol.

Embodiment 31: The device of embodiment 29, wherein the first phasesolution comprises polypropylene glycol.

Embodiment 32: The device according to any one of embodiments 29-31,wherein said salt comprises potassium phosphate, sodium sulfate,magnesium sulfate, ammonium sulfate, or sodium citrate.

Embodiment 33: The device according to any one of embodiments 29-31,wherein said salt is potassium phosphate.

Embodiment 34: The device according to any one of embodiments 2-20,wherein the first phase solution comprises a Component 1 of Table 1 andthe second phase solution comprises a Component 2 of Table 1.

Embodiment 35: The device according to any one of embodiments 1-34,wherein said device further comprises a probe that interacts with thetarget bacterium, fungus, or virus.

Embodiment 36: The device of embodiment 35, wherein the device comprisesone or more probes that interact with at least 1 target bacteria, fungior virus, or at least two different target bacteria, fungi or virus, orat least 3 different target bacteria, fungi or virus, or at least 4different target bacteria, fungi or virus, or at least 5 differenttarget bacteria, fungi or virus, or at least 7 different targetbacteria, fungi or virus, or at least 10 different target bacteria,fungi or virus, or at least 15 different target bacteria, fungi orvirus, or at least 20 different target bacteria, fungi or virus.

Embodiment 37: The device according to any one of embodiments 35-36,wherein the device includes at least two different probes, or at least 3different probes, or at least 4 different probes, or at least 5different probes, or at least 7 different probes, or at least 10different probes, or at least 15 different probes, or at least 20different probes.

Embodiment 38: The device according to any one of embodiments 35-37,wherein the probe comprises a synthetic polymer, a metal, a mineral, aglass, a quartz, a ceramic, a biological polymer, or a plastic.

Embodiment 39: The device of embodiment 38, wherein the probe comprisespolyethylene, polypropylene, cellulose, chitin, nylon, polyoxymethylene,polytetrafluoroethylene, or polyvinyl chloride.

Embodiment 40: The device of embodiment 38, wherein the probe comprisesa biological polymer comprises dextran, polypropylene, or polyethyleneglycol.

Embodiment 41: The device of embodiment 38, wherein the probe comprisesgold, silver, or platinum.

Embodiment 42: The device according to any one of embodiments 38-41,wherein the probe comprises a nanoparticle.

Embodiment 43: The device of embodiment 42, wherein the nanoparticle isa gold nanoparticle.

Embodiment 44: The device according to any one of embodiments 38-43,wherein the probe comprises a coating.

Embodiment 45: The device of embodiment 44, wherein the coatingcomprises polypropylene glycol or polyethylene glycol.

Embodiment 46: The device of embodiment 44, wherein the coatingcomprises dextran.

Embodiment 47: The device of embodiment 44, wherein the coatingcomprises a hydrophilic protein.

Embodiment 48: The device of embodiment 44, wherein the coatingcomprises serum albumin.

Embodiment 49: The device according to any one of embodiments 44-48,wherein the coating has an affinity for the first phase solution or thesecond phase solution.

Embodiment 50: The device according to any one of embodiments 35-49,wherein the probe further comprises a binding moiety that binds thetarget bacterium, fungus or virus.

Embodiment 51: The device of embodiment 50, wherein the binding moietycomprises an antibody, a lectin, a protein, a glycoprotein, a nucleicacid, a small molecule, a polymer, or a lipid.

Embodiment 52: The device of embodiment 50, wherein the binding moietyis an antibody or antibody fragment.

Embodiment 53: The device of embodiment 52, wherein said antibody is anantibody that specifically binds the bacterium, fungus, or virus.

Embodiment 54: The device according to any one of embodiments 1-53,wherein said device further comprises a signal enhancement reagent.

Embodiment 55: The device of embodiment 54, wherein said signalenhancement reagent comprises a substrate that reacts with an enzymethat is decorated on the surface of probe to form a strong visibleproduct.

Embodiment 56: The device of embodiment 55, wherein said signalenhancement comprises a silver ion.

Embodiment 57: The device according to any one of embodiments 1-56,wherein said device is configured to perform a competition assay.

Embodiment 58: The device according to any one of embodiments 1-56,wherein said device is configured to perform a sandwich assay.

Embodiment 59: The device according to any one of embodiments 1-58,wherein said device detects an analyte (e.g., a bacterium) at aconcentration of less than about 6×10⁶ cells/mL, or less than about3×10⁶ cells/ml, or less than about 1×10⁵ cells/mL, less than about 1×10⁶CFU/mL, or less than about 50 μg/mL.

Embodiment 60: The device according to any one of embodiments 1-59,wherein false positives appear at an analyte concentration of less thanabout 12 ng/μL, or less than about 10 ng/μL, or less than about 8 ng/μL,or less than about 6 ng/μL, or less than about 4 ng/μL, or less thanabout 2 ng/μL.

Embodiment 61: A kit for the detection and/or quantification of abacterium, said kit comprising: a device according to any one ofembodiments 1-60; and a collection device for collecting a biologicalsample.

Embodiment 62: The kit of embodiment 61, wherein said collection devicecomprises a device for collecting oral fluid.

Embodiment 63: The kit of embodiment 61, wherein said collection devicecomprises a device for collecting blood.

Embodiment 64: The kit of embodiment 61, wherein said collection devicecomprises a urine collection device.

Embodiment 65: The kit of embodiment 61, wherein said collection devicecomprises a device for collecting vaginal fluid or from a vaginal swabor from an endocervical swab.

Embodiment 66: The kit of embodiment 61, wherein said collection devicecomprises a device for collecting an environmental sample.

Embodiment 67: A method of detecting and/or quantifying a bacterium,fungus, or virus in a sample comprising:

-   -   i) applying the sample to the device of any one of embodiments        1-60; and    -   ii) detecting a presence or absence and/or quantifying the        bacterium fungus or virus on the LFA or detection component of        the flow-through device.

Embodiment 68: A method of detecting and/or quantifying a bacterium,fungus, or virus in a sample comprising:

-   -   i) applying the sample to an aqueous two-phase system (ATPS);    -   ii) applying the ATPS or component thereof containing the sample        to the device any one of embodiments 1-60; and    -   iii) detecting a presence or absence and/or quantifying the        bacterium on the LFA or detection component of the flow-through        device.

Embodiment 69: The method according to any one of embodiments 67-68,wherein the sample is an environmental sample, an oral sample, a vaginalfluid sample, a urine sample, a sample from a vaginal swab, or a samplefrom an endocervical swab.

Embodiment 70: The method of embodiment 69, wherein said sample is abuccal sample, or an oral fluid sample.

Embodiment 72: The method according to any one of embodiments 67-70,wherein false positives appear at an analyte concentration of less thanabout 12 ng/μL, or less than about 10 ng/μL, or less than about 8 ng/μL,or less than about 6 ng/μL, or less than about 4 ng/μL, or less thanabout 2 ng/μL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates rapid target concentration and easy extraction usingPPG/salt ATPS in a syringe. Sample containing target biomolecules(purple) is mixed with the ATPS solution. After 5-10 min of incubationat room temperature, the targets are concentrated extremely in thebottom phase, and can be easily extracted and applied to the subsequentdetection step by pressing the plunger of the syringe.

FIG. 2 illustrates an assay where the ATPS containing sample is appliedto an assay device (e.g., LFA).

FIG. 3 shows a schematic of a reaction pad demonstrating the concept ofa semi-quantitative lateral-flow assay for the detection of S. mutans.The specific antibody for S. mutans is immobilized on the test lineswith various concentrations. The number of test lines that appearcorrelate with the concentration of S. mutans in the samples, which canbe used to predict the risk of dental caries development.

FIG. 4 shows a schematic of an all-in-one spot test for the detection oftarget biomolecules. ATPS components and colorimetric indicator aredehydrated onto the concentration component and the conjugate pad,respectively. The user can simply apply the sample solution to thedevice. After which, concentration of the target biomolecules wouldoccur within the concentration component. Subsequently, the solutionwill rehydrate and bind to the colorimetric indicator on the conjugatepad, and the resulting indicator-target complexes will be captured onthe reaction pad as shown by a visible spot.

FIG. 5 illustrates the concentration and detection of S. mutans usingPPG/salt ATPS and LFA.

FIG. 6 illustrates the detection of S. mutans in plaque from 4 subjects.The higher test line intensity indicates a greater concentration of S.mutans in the subject.

FIG. 7 illustrates the detection of S. mutans in plaque before and afterbrushing teeth. The result indicated that brushing teeth is effective inremoving S. mutans and lowering the risk to develop dental caries.

FIG. 8 illustrates the detection of C. trachomatis in PBS using LFAalone and using ATPS with LFA.

FIG. 9 illustrates the performance of a device described herein comparedto an FDA approved, commercially available Chlamydia LFA in a clinicalurine sample collected from a C. trachomatis positive patient. Ourdevice is able to provide a true positive result (the presence of thetest line), while the commercial test gave a false negative result (theabsence of the test line).

FIG. 10 illustrates one embodiment of a lateral-flow assay (LFA)described herein using a sandwich format.

FIG. 11 . The integrated ARROW and LFA diagnostic design. (Left) Designlayout of the integrated ARROW and LFA. (Middle) Image of the ARROW.(Right) SEM images of the dehydrated PEG on fiberglass, blankfiberglass, and dehydrated potassium phosphate on fiberglass. The topand bottom tips of the fiberglass paper sheet are also blank fiberglass.

FIG. 12 . Demonstrating the importance of ATPS component rehydrationorder. Time-lapse visualization of phase separation within a singlesheet of the ARROW design when the rehydration order of the PEG andpotassium phosphate are switched. Close up images are shown of thedownstream region where phase separation occurred, and therefore, thefirst image is at t=6 instead of t=0. The dotted line (- - -)encompasses the region of the paper that predominantly contained thePEG-rich phase, identified by the light blue color. Visualization andidentification of the PEG-rich phase, PEG-poor phase, andmacroscopically mixed domain regions were accomplished by flowing asuspension of BSA-DGNPs and Brilliant Blue dye.

FIG. 13 . Improvement in the limit of detection of C. trachomatis LFA byincorporation of the ARROW. Comparison of LFA results at varying C.trachomatis concentrations, with and without the ARROW. Test lines arelocated on the bottom of the LFA strips while the control lines arelocated on the top of the LFA test strips. Negative control results areshown in the left most panels for 0 ng/μL C. trachomatis.

FIG. 14 . Quantification of test line intensities. Plot of thequantified LFA test line intensities for the ARROW and LFA system andthe LFA only system.

FIG. 15 . Representative result (sample #4, Table 2) of the head-to-headcomparison between QuickVue, Phase's LFA, and Phase's LFA+ATPS. Thepresence of the test line (T) indicates a true positive result. Only ourLFA+ATPS had a visible test line indicating a true positive result.

DETAILED DESCRIPTION

In various embodiments methods and devices are provided for analytecollection, extraction, concentration, and detection for clinicalapplications. In certain embodiments the devices permit the rapiddetection and/or quantification of bacteria, fungi, and viruses inbiological samples (e.g., oral, urine, and vaginal samples).

In certain embodiments lateral-flow assay (LFA) devices (see, e.g., FIG.10 ) and/or flow-through (spot) assay devices (see, e.g., FIG. 4 ) areprovided that are accurate, sensitive, portable, disposable, and wellsuited to use at point of care with minimal training or equipment.

In certain embodiments the lateral-flow assay devices or theflow-through assay devices can be used directly with a sample to beassayed. In certain embodiments the lateral-flow assay devices or theflow-through assay devices can be used with a sample in which the target(e.g. target molecule(s), target microorganism(s), etc.) have beenconcentrated before application to the device, using for example, anaqueous two-phase system (ATPS). In certain embodiments the target (e.g.target molecule(s), target microorganism(s), etc.) are concentrated,using e.g., ATPS, on the device itself.

Concentration of the Target Biomolecules

The concentration of target biomolecules using ATPS can be performed ineither a bulk liquid, or as the sample solution flows in, e.g., alateral-flow assay or a flow-through (spot assay), e.g., in a papermembrane.

Concentration in Liquid ATPS

In certain illustrative embodiments a collected sample, (e.g., a tissuesample, a biological fluid such as urine, saliva, and blood, sputum,vaginal fluid, seminal fluid, cerebrospinal fluid, lymph, vaginal swab,endocervical swab, plaque from teeth, and the like), can be combinedwith a suspending solution (e.g., a buffer) or combined directly with anATPS solution or directly applied to paper or a suspending solutioncontaining the sample applied to a paper to rehydrate ATPS componentsthat were previously dried onto paper. In some cases, mixing by the usermay be required to achieve a well-mixed, homogeneous solution. Invarious embodiments a polymer/salt, polymer/polymer, micellar/polymer,or micellar ATPS may be used. In one of the examples described below, apolypropylene glycol (PPG):potassium phosphate salt ATPS was used toconcentrate Streptococcus mutans (S. mutans) by 60-fold within 10 min(see, e.g., FIG. 1 ). If the target analyte (e.g., target biomolecule)is large, such as a bacterium, fungus or virus, it will be partitioned,or distributed, extremely into one of the two phases in the ATPS, whichcan then be introduced to a downstream detection component in the LFA orflow-through assay. In certain embodiments, if the target analyte issmall, such as a protein, metabolite, hormone, large probes that aredecorated with specific binding moieties can be used to capture thetarget, and subsequently be concentrated into one of the phases in ATPSfor downstream detection. In certain embodiments the phase that containsthe concentrated target analyte(s) (e.g., biomolecule(s)) can beintroduced to the detection component by physical extraction using apipette or dropper, or can be introduced via a syringe, e.g., asillustrated in FIG. 1 .

Concentration as Fluid Flows on Paper

In various embodiments the concentration step can also be acceleratedwith paper. For example, the collected specimen can be mixed with ATPScomponents and introduced to a paper device that can facilitate,enhance, and accelerate phase separation. The target biomolecules can beconcentrated in the leading front of the flow on the paper membrane andcan seamlessly be introduced to the subsequent detection component.

Alternatively, the ATPS components can be pre-dehydrated onto the papermembranes. In this case, the collected specimen can be directly appliedto the paper membrane without pre-mixing with the ATPS components.

Detection of Target Biomolecules

In various embodiments the detection components in the assay systemscontemplated herein can be paper-based detection components. In certainembodiments the paper-based detection component (can be in the form of alateral-flow test strip (see, e.g., FIGS. 3 and 10 ) or a flow-throughdevice (spot test) (see, e.g. FIG. 4 ). In various embodiments both formfactors may contain, but are not limited to, one or more of thefollowing components:

Sample Pad

In certain embodiments a sample pad, when present, can connect theconcentration component to the detection component. It can act as afilter that can remove debris, contaminants, and mucus from thecollected fluid. It can also store dried reagents, and when rehydrated,these reagents can (i) adjust the solution for optimal detectionconditions (pH, ionic strength, etc); and (ii) break down mucus,glycoproteins, and other viscous materials in the collected specimenthat may affect detection. Illustrative materials for the sample padinclude, but are not limited to, cellulose, nitrocellulose, fiberglass,cotton, woven or nonwoven paper, etc. Reagents on the pad may include,but are not limited to, surfactants such as Triton X-100, Tween 20, orsodium dodecyl sulfate, etc.; polymers such as polyethylene glycol,poloxamer, polyvinylpyrrolidone (PVP), etc.; buffers such asphosphate-buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (HEPES), Tris(hydroxymethyl)aminomethane (Tris), sodium borate,TRICINE, etc.; proteins such as albumin, etc.; enzymes such as protease,etc.; salts such as sodium chloride, sodium phosphate, sodium cholate,potassium phosphate, etc. In various embodiments these reagents can beapplied to the sample pad by (i) soaking the paper material in thereagent solution, or (ii) through wicking the membrane via capillaryflow. The treated sample pad can be dried by (i) air drying (let sit atroom temperature); (ii) baking (place in high temperature using an ovenor heating device); (iii) vacuum; or (iv) lyophilization.

Conjugate Pad

In various embodiments a conjugate pad, when present can containdehydrated colorimetric indicators decorated with binding moieties thatbind the target analyte(s). In certain embodiments the binding moietiesare specific binding moieties that have high affinity towards the targetanalyte(s) (e.g., bacterium, fungus, virus, etc.). When the samplesolution reaches the conjugate pad, the colorimetric indicators arerehydrated. The binding moieties on the colorimetric indicators can thenbind to the target analyte(s) and the resulting complexes can flow tothe reaction pad. In certain embodiments the colorimetric indicators cancomprise metallic particles such as gold, silver particles, polymericparticles such as latex beads, and polystyrene particles encapsulatingvisible or fluorescent dyes. Illustrative materials material for theconjugate pad include, but are not limited to, cellulose,nitrocellulose, fiberglass, cotton, woven or nonwoven paper etc. Incertain embodiments the colorimetric indicators can be applied anddehydrated onto the pad as described above.

Reaction Pad

In certain embodiments the reaction pad, when present, can compriseimmobilized reagents, and when the immobilized reagents react with thesample solution, they may produce signals (e.g., visual signals) toindicate the presence or absence or quantity of the target analyte(s).Illustrative materials for the reaction pad include, but are not limitedto cellulose, nitrocellulose, fiberglass, cotton, woven or nonwovenpaper etc.

Lateral-Flow Format

In certain embodiments for a lateral-flow test strip, the reagents onthe reaction pad will be immobilized in the form of lines perpendicularto the direction of flow to ensure all samples can interact with theimmobilized reagents. The concentrations of the reagents can beoptimized to control the signal intensities, and thus, control thesensitivity of the assay. For example, a semi-quantitative assay can bedesigned by immobilizing multiple lines of the same reagent with variousconcentrations. Each line therefore will yield signals only when aspecific concentration of target biomolecules is reached. Theconcentration of the target biomolecules can then be interpreted bycounting the number of lines that are visible (see, e.g., FIG. 3 ). Forthe detection of S. mutans, this semi-quantitative assay mayparticularly be useful to provide better prediction of dental cariessince S. mutans concentration is highly correlated to the risk ofdeveloping dental caries.

In addition, multiple lines of different reagents can be immobilized onthe same strip to detect multiple target analyte(s). This allows thedevelopment of multiplex assays.

Flow-Through Format

In certain embodiments, e.g., for a flow-through test, instead of lines,the reagents can be immobilized on the entire reaction pad. If thetarget analyte is present, it will bind to the colorimetric indicator onthe conjugate pad and be trapped on the reaction pad as theindicator-target complex binds to the immobilized reagent. A visiblespot will therefore appear if the target biomolecule is present. Thistest can be used if the sample volume is too low to wick up alateral-flow test strip. The color intensity of the visible spot iscorrelated to the concentration of target analyte(s) (e.g.,biomolecules), while the size of the spot is correlated to the samplevolume. In certain embodiments the concentration component can be placeddirectly on top of the flow-through test to remove the need forextracting and applying the concentrated samples to the detectioncomponent (see, e.g., FIG. 4 ).

In various embodiments the immobilized reagents can comprise a specificantibody against the target analyte (primary antibody), antibodiesagainst the primary antibody (secondary antibody), antigens, proteins,or antigen-protein conjugates. Illustrative materials for the reactionpad include, but are not limited to cellulose, nitrocellulose,fiberglass, cotton, woven and nonwoven paper etc. In various embodimentsthe reagents can be applied and dehydrated onto the pad as describedabove.

Sink

In certain embodiments the sink, when present, can comprise an absorbentpad that collect excess fluid and prevents back-flow which can affectthe test performance. Illustrative materials for the sink include, butare not limited to cellulose, nitrocellulose, fiberglass, cotton, wovenand nonwoven paper etc.

Signal Enhancement

In various embodiments the visible signal intensity can be enhanced toimprove the accuracy of the detection assay. This can be performed byintroducing additional reagents to the reaction pad after the initialdetection assay. In certain embodiments the signal enhancement reagentcan comprise a substrate that reacts with an enzyme that is decorated onthe surface of, e.g., colorimetric indicator to form a strong visibleproduct. By way of example, if the colorimetric indicator comprises agold probe, the signal enhancement can be achieved by silver-enhancementlabeling, where an enhancement reagent containing silver ion can beapplied to the reaction pad where the gold probe is bound to theimmobilized line/spot. In this scenario, the gold probes can act asnucleation sites so that silver can be deposited onto the particle,resulting in increased signal intensity. In these examples, the signalenhancement reagents can either be added separately after the initialdetection assay, or stored/dehydrated on the paper device to be releasedautomatically/manually.

The foregoing components and assay formats are illustrative andnon-limiting. Using the teachings and examples, provided herein,numerous other assay devices and configurations will be available to oneof skill in the art and some further design considerations andcomponents are described below.

Lateral-Flow Assay (LFA) or Flow-Through (Spot) Assay

As explained above, in certain embodiments, the devices and systemsdescribed herein are configured to provide a lateral-flow assay (LFA) ora flow-through (spot) assay for detection of the target analyte in asample, where the LFA or spot assay is used alone or in conjunction withan aqueous two-phase system (ATPS). In some embodiments, the LFA or spotassay comprises a porous matrix into which is disposed the ATPS orcomponents thereof, where the porous matrix is configured to and hasporosity sufficient to allow the ATPS or components thereof toflow-through the porous matrix when the ATPS or components thereof arein a fluid phase. Such porous LFA or spot assay devices are referred toherein as paper or paper fluidic devices and these terms are usedinterchangeably.

The term “paper”, as used herein, is not limited to thin sheets from thepulp of wood or other fibrous plant substances although, in certainembodiments the use of such papers in the devices described herein iscontemplated. Papers more generally refer to porous materials often insheet form, but not limited thereto that allow a fluid to flow-through.

In some embodiments, the porous matrix is sufficiently porous to allowthe mixed phase solution, first phase solution and/or second phasesolution of the ATPS, and/or target analyte, to flow-through the LFA orflow-through assay. In some embodiments, the porous matrix issufficiently long and/or deep enough for the mixed phase solution, firstphase solution and/or second phase solution, and/or target analyte, toflow vertically and/or horizontally through the LFA or flow-through(spot) assay device. In some embodiments, the first phase solution flowsthrough the porous matrix at a first rate and the second phase solutionflows through the porous matrix at a second rate, where the first rateand the second rate are different. In some embodiments of the LFA orspot assay the porous matrix comprises inter alia a material such as asintered glass ceramic, a mineral, cellulose, a fiberglass, anitrocellulose, polyvinylidene fluoride, a nylon, a charge modifiednylon, a polyethersulfone, combinations thereof, and the like.

Concentrate-as-it-Flows

It was discovered that ATPSs can phase separate as the solution flowsthrough a porous substrate (e.g., a paper) which we have termed“concentrate-as-it-flows”. Moreover, it was also discovered that flowthrough the paper significantly speeds up the concentration process.Based this phenomenon, the lateral-flow assay devices and theflow-through assay devices described herein can comprise a paper fluidiccomponent that fully integrates the necessary components for a combinedATPS concentration with the LFA or flow-through detection. It wasdiscovered that when a mixed ATPS solution is applied to certain papermaterials, phase separation and analyte concentration occur as thesolution flows. We also demonstrated that this phenomenon is preservedeven when making an ATPS that had varying volume ratios, e.g., volume ofthe top phase divided by that of the bottom phase.

In some embodiments, the LFA or the spot assay (e.g., the concentrationcomponent of the spot assay) comprises a paper. In some embodiments, thepaper comprises a sheet of porous material that allows fluid toflow-through it. In some embodiments, the paper comprises a plurality ofsheets of porous material that allows fluid to flow-through them (e.g.,as illustrated in FIG. 4 ). In some embodiments, the paper comprises oneor more materials such as cellulose, fiberglass, nitrocellulose,polyvinylidine fluoride, charge modified nylon, polyether sulfone, andthe like. In some embodiments, the paper is a HI-FLOW PLUS® membrane.

In some embodiments, the paper is a woven paper. In some embodiments,the paper is a Whatman paper. In some embodiments, the Whatman papercomprises Whatman 517, Whatman MF1, Whatman VF1, Whatman Fusion 5,Whatman GF/DVA, Whatman LF1, Whatman CF1, and/or Whatman CF4.

In some embodiments, the paper concentrates the target analyte as thetarget analyte flows through the LFA or through the concentrationcomponent of a flow-through assay (e.g. a“concentrate-as-it-flows”-based device). In some embodiments, the paperconcentrates the target analyte as the target analyte flows through theLFA horizontally. In some embodiments, the paper concentrates the targetanalyte as the target analyte flows through the LFA or flow-throughassay vertically.

In some embodiments, the paper has a property that influences whichphase solution will become the “leading fluid.” By way of non-limitingexample, when using a PEG-salt ATPS, adding the solution to fiberglasspaper will cause the salt phase to become the leading solution, whileusing cellulose paper will cause the PEG phase to become the leadingsolution. In some embodiments, phase separation within the paperaccelerates phase separation. Also by way of non-limiting example, amicelle ATPS typically takes several hours to phase separate in astagnant ATPS, but if applied to a paper strip, this phase separationoccurs in minutes. This speeds up the diagnostic process by allowing theATPSs, which are traditionally the rate-determining step in the process,to become more viable options for our rapid paper diagnostic assays. Insome embodiments, the ‘concentrate-as-it-flows’ device comprises aPEG-salt ATPS (e.g., as illustrated in the Examples). In someembodiments, the ‘concentrate-as-it-flows’ device comprises a micellarATPS. In some embodiments, the LFA device or the flow-through assaydevice comprises fiberglass paper or nitrocellulose paper.

In certain embodiments the LFA or flow-through assay device comprises afilter that removes debris (e.g., blood cells or other particulates), asample pad where the sample comprising the target analyte is applied tothe device, a detection zone (e.g. test line and control line) wherethere the target analyte binds and is detected, and an absorbent pad(e.g., a dry receiving paper) that can absorb excess sample and/orsolutions applied to the LFA or flow-through device (see, e.g., FIG. 10). In some embodiments, the control line and/or test line is not a lineper se, but a region or spot.

In some embodiments, the LFA comprises an LFA strip. The terms “LFA” and“LFA strip” are used interchangeably herein. In some embodiments, theLFA strip has a length greater than its width and depth. In someembodiments, the LFA is rectangular. In some embodiments, the LFA has ashape that is round, ovoid, square, polygonal, or irregular-shaped. Insome embodiments, the LFA comprises a plurality of routes and/orjunctions. In some embodiments, the LFA strip comprises the sample pad,detection zone and absorbent pad. In some embodiments, the detectionzone is located between the sample pad and the absorbent pad, theabsorbent pad wicking the sample with the target analyte away from thesample pad and toward the detection zone.

Sandwich Assay

In some embodiments, the LFA or flow-through (spot) assay device isconfigured to provide or run a sandwich assay (see e.g., FIG. 1 , bottomleft, in copending PCT Application No: PCT/US2015/019297, filed on Mar.6, 2015, which is hereby incorporated by reference for the LFAconfigurations described therein). In some embodiments, the sandwichassay comprises a capture moiety that binds the target analyte. In someembodiments, the device comprises a probe. In some embodiments, theprobe comprises a detectable property (colorimetric, fluorescent,radioactive, etc.). In some embodiments, the probe comprises a bindingmoiety that interacts with the target analyte (e.g. an antibody). Insome embodiments, the probe is added to the sample and binds the targetanalyte to form a probe-analyte complex.

Competition Assay

In some embodiments, the LFA comprises a competition assay. In someembodiments, the probe is added to the sample and binds the targetanalyte to form a probe-analyte complex. In some embodiments, the LFAcomprises the target analyte immobilized on the test line. In someembodiments, the probe is saturated by the target analyte in the sampleand the probe will not bind to the target analyte immobilized on thetest line. In some embodiments, the absence of the detectable signal onthe test line indicates a positive result. In some embodiments, there isno target analyte present in the sample, and the probe binds to thetarget analyte on the test line, indicating a negative result. In someembodiments, the LFA comprises a probe capture moiety on a control linethat interacts directly with the probe, and regardless of the presenceof the target analyte in the sample, the probe can bind to the probecapture moiety and accumulate on the control line. In some embodiments,the probe becomes immobilized and detected on the control line,indicating a valid test. In some embodiments, a positive result (e.g.,target analyte is present in sample) is indicated by the absence of adetectable signal at the test line and the presence of a detectablesignal at the control line. In some embodiments, a negative result isindicated by a detectable signal at both the test and control lines.

In some embodiments of a sandwich format assay, the probe-analytecomplex is applied to the sample pad and flows through the LFA orthrough the flow-through device towards the absorbent pad. In someembodiments, the target analyte of the probe-analyte complex binds tothe capture moiety. In some embodiments, the capture moiety isimmobilized on a test line or a test region (e.g., a test layer in aflow-through device) and the probe-analyte complex becomes immobilizedon the test line or in the test region. In some embodiments, the probeis colorimetric, and the test line or test region will exhibit a strongcolor (e.g. detectable signal) as the probe-analyte complex accumulatesat the test line or in the test region, indicating a positive result. Insome embodiments, there is no target analyte present in the sample, andthe probe of the probe-analyte complex does not interact with thecapture moiety, and the absence of the test line or signal in the testregion indicates a negative result. In some embodiments, the LFAcomprises a probe capture moiety on a control line (or in a controlregion, e.g., of a flow-through assay device) that interacts directlywith the probe and/or the binding moiety, and thus, regardless of thepresence of the target analyte in the sample, the probe/binding moietybinds to the probe capture moiety and accumulate on the control line orin the control region. In some embodiments, the probe capture moiety isa secondary antibody that binds the binding moiety, wherein the bindingmoiety is a primary antibody that binds that target analyte. In someembodiments, the probe becomes immobilized and detected on the controlline or in the control region, indicating a valid test. In someembodiments, a positive result (e.g. target analyte is present insample) is indicated by a detectable signal at the test line (or testregion) and the control line (or control region). In some embodiments, anegative result is indicated by a detectable signal at the control lineor in the control region.

Aqueous Two-Phase System (ATPS)

In certain embodiments the devices described herein are configured towork in conjunction with an aqueous two-phase system (ATPS), e.g., in asyringe or other vessel, or they are configured to support an aqueoustwo-phase system (ATPS). In some embodiments, the ATPS comprises a phasesolution. The term “phase solution” generally refers to a first phasesolution or a second phase solution of the ATPS. In some embodiments,the phase solution is in a mixed solution (e.g. with the first/secondphase solution). In some embodiments, the phase solution is thefirst/second phase solution after it separates from the mixed solutionof the ATPS. In some embodiments, the phase solution is the first/secondphase solution after it separates from the mixed solution in the LFA orflow-through assay. In certain embodiments the phase solution can referto the second phase solution while it is in a mixed state (e.g. with thefirst phase solution). In some embodiments, the phase solution is aleading fluid in the LFA or flow-through assay. In some embodiments, thephase solution is a lagging fluid in the LFA or flow-through assay.

In some embodiments, the ATPS comprises two aqueous solutions, a firstphase solution and a second phase solution that are initially mixed(e.g., a mixed phase solution). In some embodiments, the mixed phasesolution is a homogeneous solution, while in certain other embodimentsthe first phase solution and the second phase solution are immiscible.In some embodiments, the first phase solution and the second phasesolution are immiscible, but domains of the first phase solution aremixed with domains of the second phase solution. In some embodiments,the immiscibility is driven by changes in temperature, and/or changes inthe concentrations of the different components, such as salt. In someembodiments, the first/second phase solutions comprise components, suchas, micelles, salts, and/or polymers. In some embodiments, the targetanalyte (e.g., biomolecule, bacterium (or fragment thereof), fungus (orfragment thereof), or virus, and the like) in contact with the ATPS,distributes, partitions, and/or concentrates preferentially into thefirst phase solution over the second phase solution, or vice versa,based on its physical and chemical properties, such as size, shape,hydrophobicity, and charge. In some embodiments, the target analyte(e.g. a bacterium, fungus, virus, etc.) partitions predominantly (orextremely) into the first or second phase solution of the ATPS, andtherefore concentrates in the ATPS. In some embodiments, the targetanalyte is concentrated by adjusting the ratio of volumes between thefirst phase solution and the second phase solution. In some embodiments,the target analyte is concentrated by reducing the volume of the phasein which the analyte partitions. By way of illustration, in someembodiments, the target analyte is concentrated by 10-fold in the firstphase solution, e.g., by using a 1:9 volume ratio of first phasesolution to second phase solution, since the volume of the phase intowhich the analyte extremely partitions into is 1/10 the total volume.

In some embodiments, other concentrations are obtained by using otherratios. Thus, in some embodiments the ratio of the first phase solutionto the second phase solution comprises a ratio of about 1:1, about 1:2,about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about1:9, or about 1:10. In some embodiments the ratio of the first phasesolution to the second phase solution comprises a ratio of about 1:20,about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80,about 1:90, or about 1:100. In some embodiments the ratio of the firstphase solution to the second phase solution comprises a ratio of about1:200, about 1:300, about 1:400, about 1:500, about 1:600, about 1:700,about 1:800, about 1:900, or about 1:1000.

In some embodiments the ratio of the second phase solution to the firstphase solution comprises a ratio of about 1:1, about 1:2, about 1:3,about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, orabout 1:10. In some embodiments the ratio of the second phase solutionto the first phase solution comprises a ratio of about 1:20, about 1:30,about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90,or about 1:100. In some embodiments the ratio of the second phasesolution to the first phase solution comprises a ratio of about 1:200,about 1:300, about 1:400, about 1:500, about 1:600, about 1:700, about1:800, about 1:900, or about 1:1000.

In some embodiments, the analyte partitions substantially evenly betweenthe first phase solution and second phase solution, preventingconcentration of the analyte. In such systems, concentration of thetarget analyte can be achieved by introducing an additional component,such as a probe that captures the target analyte, and wherein the probepartitions predominantly into one phase, thereby enhancing thepartitioning behavior of the target analyte to enable concentration. Insome embodiments, the first/second phase solution containing theconcentrated analyte is collected and applied to the LFA or to theflow-through assay device.

In some embodiments, the first/second phase solution comprises amicellar solution. In some embodiments, the micellar solution comprisesa nonionic surfactant. In some embodiments, the micellar solutioncomprises a detergent. In some embodiments, the micellar solutioncomprises Triton-X. In some embodiments, the micellar solution comprisesa polymer similar to Triton-X, such as Igepal CA-630 and Nonidet P-40,and the like, by way of non-limiting example. In some embodiments, themicellar solution consists essentially of Triton-X.

In some embodiments, the micellar solution has a viscosity (at roomtemperature (˜25° C.) of about 0.01 centipoise to about 5000 centipoise,about 0.01 centipoise to about 4500 centipoise, about 0.01 centipoise toabout 4000 centipoise, about 0.01 centipoise to about 3500 centipoise,about 0.01 centipoise to about 3000 centipoise, about 0.01 centipoise toabout 2500 centipoise, about 0.01 centipoise to about 2000 centipoise,about 0.01 centipoise to about 1500 centipoise, about 0.01 centipoise toabout 1000 centipoise, or about 0.01 centipoise to about 500 centipoise.In some embodiments, the micellar solution has a viscosity at roomtemperature of about 0.01 centipoise to about 450 centipoise, about 0.01centipoise to about 400 centipoise, about 0.01 centipoise to about 350centipoise, about 0.01 centipoise to about 300 centipoise, about 0.01centipoise to about 250 centipoise, about 0.01 centipoise to about 200centipoise, about 0.01 centipoise to about 150 centipoise, or about 0.01centipoise to about 100 centipoise.

In some embodiments, the first/second phase solution comprises a polymer(e.g., polymer solution). In certain embodiments, the polymer is apolyethylene glycol (PEG). In various embodiments, the PEG may have amolecular weight between 1000 and 100,000. In certain embodiments, thePEG comprises PEG-4600, PEG-8000, or PEG-20,000. In certain embodiments,the polymer is polypropylene glycol (PPG). In various embodiments, thePPG may have a molecular weight between 100 and 10,000. In certainembodiments, the PPG comprises PPG 425. In certain embodiments, thepolymer is dextran. In various embodiments, the dextran may have amolecular weight between 1000 and 1,000,000. In certain embodiments, thedextran comprises dextran 6000, dextran 9000, dextran-35,000, ordextran-200,000.

In some embodiments, the polymer solution comprises a polymer solutionthat is about 0.01% w/w polymer, or about 0.05% w/w polymer, or about0.1% w/w polymer, or about 0.15% w/w polymer, or about 0.2% w/w polymer,or about 0.25% w/w polymer, or about 0.3% w/w polymer, or about 0.35%w/w polymer, or about 0.4% w/w polymer, or about 0.45% w/w polymer, orabout 0.5% w/w polymer, or about 0.55% w/w polymer, or about 0.6% w/wpolymer, or about 0.65% w/w polymer, or about 0.7% w/w polymer, or about0.75% w/w polymer, or about 0.8% w/w polymer, or about 0.85% w/wpolymer, or about 0.9% w/w polymer, or about 0.95% w/w polymer, or about1% w/w polymer. In some embodiments, the polymer solution comprises apolymer solution that is about 1% w/w polymer, or about 2% w/w polymer,or about 3% w/w polymer, or about 4% w/w polymer, or about 5% w/wpolymer, or about 6% w/w polymer, or about 7% w/w polymer, or about 8%w/w polymer, or about 9% w/w polymer, or about 10% w/w polymer, or about110% w/w polymer, or about 12% w/w polymer, or about 13% w/w polymer, orabout 14% w/w polymer, or about 15% w/w polymer, or about 16% w/wpolymer, or about 17% w/w polymer, or about 18% w/w polymer, or about19% w/w polymer, or about 20% w/w polymer, or about 21% w/w polymer, orabout 22% w/w polymer, or about 23% w/w polymer, or about 24% w/wpolymer, or about 25% w/w polymer, or about 26% w/w polymer, or about27% w/w polymer, or about 28% w/w polymer, or about 29% w/w polymer, orabout 30% w/w polymer, or about 31% w/w polymer, or about 32% w/wpolymer, or about 33% w/w polymer, or about 34% w/w polymer, or about35% w/w polymer, or about 36% w/w polymer, or about 37% w/w polymer, orabout 38% w/w polymer, or about 39% w/w polymer, or about 40% w/wpolymer, or about 41% w/w polymer, or about 42% w/w polymer, or about43% w/w polymer, or about 44% w/w polymer, or about 45% w/w polymer, orabout 46% w/w polymer, or about 47% w/w polymer, or about 48% w/wpolymer, or about 49% w/w polymer, or and about 50% w/w polymer. In someembodiments, the polymer solution comprises a polymer solution that isabout 10% w/w polymer, or about 20% w/w polymer, or about 30% w/wpolymer, or about 40% w/w polymer, or about 50% w/w polymer, or about60% w/w polymer, or about 70% w/w polymer, or about 80% w/w polymer, orabout 90% w/w polymer. In some embodiments, the polymer solutioncomprises a polymer solution that is about 10% w/w polymer to about 80%w/w polymer. In some embodiments, the polymer solution comprises apolymer solution that is about 10% w/w to about 25% w/w polymer.

In some embodiments, the first and/or second phase solution comprises asalt and thereby forms a salt solution. In some embodiments, the targetanalyte (e.g., bacterium, fungus, virus, etc.) and/or a probe-analytecomplex partitions into the salt solution. In certain embodiments thesalt solution comprises a kosmotropic salt. In some embodiments the saltsolution comprises a chaotropic salt. In some embodiments, the saltcomprises one or more of a magnesium salt, a lithium salt, a sodiumsalt, a potassium salt, a cesium salt, a zinc salt, and an aluminumsalt. In some embodiments, the salt comprises a bromide salt, an iodidesalt, a fluoride salt, a carbonate salt, a sulfate salt, a citrate salt,a carboxylate salt, a borate salt, or a phosphate salt. In someembodiments, the salt is potassium phosphate. In some embodiments, thesalt is ammonium sulfate.

In some embodiments, the salt solution comprises a salt solutioncomprising about 0.01% w/w salt, or about 0.05% w/w salt, about 0.1% w/wsalt, or about 0.15% w/w salt, or about 0.2% w/w salt, or about 0.25%w/w salt, or about 0.3% w/w salt, or about 0.35% w/w salt, or about 0.4%w/w salt, or about 0.45% w/w salt, or about 0.5% w/w salt, or about0.55% w/w salt, or about 0.6% w/w salt, or about 0.65% w/w salt, orabout 0.7% w/w salt, or about 0.75% w/w salt, or about 0.8% w/w salt, orabout 0.85% w/w salt, or about 0.9% w/w salt, or about 0.95% w/w salt,or about or about 1% w/w salt. In some embodiments, the salt solutioncomprises a salt solution that is about 1% w/w salt, or about 2% w/wsalt, or about 3% w/w salt, or about 4% w/w salt, or about 5% w/w salt,or about 6% w/w salt, or about 7% w/w salt, or about 8% w/w salt, orabout 9% w/w salt, or about 10% w/w salt, or about 11% w/w salt, orabout 12% w/w salt, or about 13% w/w salt, or about 14% w/w salt, orabout 15% w/w salt, or about 16% w/w salt, or about 17% w/w salt, orabout 18% w/w salt, or about 19% w/w salt, or about 20% w/w salt, orabout 21% w/w salt, or about 22% w/w salt, or about 23% w/w salt, orabout 24% w/w salt, or about 25% w/w salt, or about 26% w/w salt, orabout 27% w/w salt, or about 28% w/w salt, or about 29% w/w salt, orabout 30% w/w salt, or about 31% w/w salt, or about 32% w/w salt, orabout 33% w/w salt, or about 34% w/w salt, or about 35% w/w salt, orabout 36% w/w salt, or about 37% w/w salt, or about 38% w/w salt, orabout 39% w/w salt, or about 40% w/w salt, or about 41% w/w salt, orabout 42% w/w salt, or about 43% w/w salt, or about 44% w/w salt, orabout 45% w/w salt, or about 46% w/w salt, or about 47% w/w salt, orabout 48% w/w salt, or about 49% w/w salt, or and about 50% w/w. In someembodiments, the salt solution comprises a salt solution that is about0.1% w/w to about 10%. In some embodiments, the salt solution is about1% w/w to about 10%.

In some embodiments, the first/second phase solution comprises a solventthat is immiscible with water. In some embodiments, the solventcomprises anon-polar organic solvent. In some embodiments, the solventcomprises an oil. In some embodiments, the solvent comprises pentane,cyclopentane, benzene, 1,4-dioxane, diethyl ether, dichloromethane,chloroform, toluene, or hexane.

In some embodiments, the first phase solution comprises a micellarsolution and the second phase solution comprises a polymer. In someembodiments, the second phase solution comprises a micellar solution andthe first phase solution comprises a polymer. In some embodiments, thefirst phase solution comprises a micellar solution and the second phasesolution comprises a salt. In some embodiments, the second phasesolution comprises a micellar solution and the first phase solutioncomprises a salt. In some embodiments, the micellar solution is aTriton-X solution. In some embodiments, the first phase solutioncomprises a first polymer and the second phase solution comprises asecond polymer. In some embodiments, the first/second polymer comprisespolyethylene glycol and/or dextran. In some embodiments, the first phasesolution comprises a polymer and the second phase solution comprises asalt. In some embodiments, the second phase solution comprises a polymerand the first phase solution comprises a salt. In some embodiments, thefirst phase solution comprises polyethylene glycol and the second phasesolution comprises potassium phosphate. In some embodiments, the secondphase solution comprises polyethylene glycol and the first phasesolution comprises potassium phosphate. In some embodiments, the firstphase solution comprises a salt and the second phase solution comprisesa salt. In some embodiments, the first phase solution comprises akosmotropic salt and the second phase solution comprises a chaotropicsalt. In some embodiments, the second phase solution comprises akosmotropic salt and the first phase solution comprises a chaotropicsalt.

In some embodiments, the first phase solution comprises a Component 1 ofTable 1 and the second phase solution comprises a Component 2 ofTable 1. In some embodiments, the second phase solution comprises aComponent 1 of Table 1 and the second phase solution comprises aComponent 2 of Table 1.

In some embodiments, the components of Table 1 are suspended ordissolved in a buffer. In some embodiments, the components of Table 1are suspended/dissolved in a buffer compatible with a biological systemfrom which the sample was derived. In some embodiments, the componentsof Table 1 are suspended/dissolved in a saline solution. In someembodiments, the components of Table 1 are suspended/dissolved in PBS.In some embodiments, the components of Table 1 are suspended/dissolvedin water.

TABLE 1 Illustrative aqueous two-phase extraction/concentration systems.Component 1 Component 2 Polymer/polymer Systems Polyethylene glycolDextran Ficoll Polyvinyl pyrrolidone Polyvinyl alcohol Hydroxypropylstarch Polypropylene glycol Dextran Hydroxypropyl dextran Polyvinylpyrrolidone Polyvinyl alcohol Dextran Hydroxypropyl dextran Polyvinylpyrrolidone Dextran Maltodextrin Methyl cellulose Dextran Hydroxypropyldextran Ethylhydroxyethyl Dextran cellulose Polymer/salt SystemsPolyethylene glycol Potassium phosphate Sodium sulfate Magnesium sulfateAmmonium sulfate Sodium citrate Propylene glycol (PPG) Potassiumphosphate Methoxypolyethylene Potassium phosphate glycol Polyvinylpyrrolidone Potassium phosphate

As illustrated in the Examples, in certain embodiments the ATPScomprises a polymer/salt ATPS. It was discovered that an ATPS comprisingpolyethylene glycol and a salt or polypropylene glycol and a saltprovides a rapid, sensitive, and accurate analytedetection/quantification.

In some embodiments, the devices described herein (e.g., an LFA or aflow-through assay device) can further comprise a collector configuredto be placed in contact with the ATPS, wherein the target analytepartitions at an interface of the collector and the first phase solutionand/or second phase solution. In some embodiments, the collectorcomprises a material that is a plastic, a mesoporous material, a silica,a polypropylene, a magnet, a magnetic particle, a paramagnetic particle,a material with a pore, a material with a groove, and/or any combinationthereof. In some embodiments, the collector comprises polypropylene. Insome embodiments, collector is optimized to increase target analytecollection. In some embodiments, the collector comprises a pore tomaximize the surface area. In some embodiments, the width of the pore isabout 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm,about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100 μm. In someembodiments, the width of the pore is about 100 μm, about 200 μm, about300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about800 μm, about 900 μm, or about 1 mm. In some embodiments, the depth ofthe pore is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm,about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100μm. In some embodiments, the depth of the pore is about 100 μm, about200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about700 μm, about 800 μm, about 900 μm, or about 1 mm.

Dehydrated ATPS in LFA or Flow-Through (Spot) Assay Device.

In some embodiments, the ATPS or components thereof are dehydrated onand/or in at least a first portion of the porous matrix comprising anLFA or in the concentration component of a flow-through assay device. Insome embodiments, application of the sample to the device hydrates theATPS, thereby converting the ATPS or components thereof to a fluidphase. Dehydration may make the device more user friendly as the userjust needs to add the sample (e.g., saliva, blood, urine, vaginal fluid,seminal fluid, sputum, cerebrospinal fluid, lymph, or similar fluid) tothe device. In some embodiments, a user only has to apply a solution ofthe sample to the strip to detect the presence/absence of the targetanalyte or to quantify the analyte. In some embodiments, the solution ofthe sample flows through the LFA or the flow-through device and the ATPSis re-solubilized, triggering phase separation within the LFA orflow-through device and subsequent concentration of the target analyte.

In some embodiments, all the necessary components for a given ATPS aremixed to form a mixed solution, applied to the paper comprising thedevice (e.g., LFA or flow-through (spot) assay), and then dehydrated.When the sample solution is added to the dehydrated paper, the ATPScomponents are rehydrated as the sample flows, resulting in phaseseparation. In some ATPSs where the phase containing the concentratedanalyte is less viscous, that phase will flow faster and theconcentrated analyte will emerge in the leading fluid and will reach thedetection zone of the LFA or flow-through assay to initiate detection.Additionally, the dehydrated ATPS component segment length (orthickness) and concentration can be adjusted for different applications.

In some embodiments, both (all) components of the ATPS are dehydrated onthe LFA or in the flow-through assay (e.g., in the separationcomponent). In some embodiments, a first ATPS component is dehydrated on(or in) the LFA or in the flow-through assay. In some embodiments, asecond ATPS component is dehydrated on or in the LFA or flow-throughassay. In some embodiments, the first phase solution component and/orfirst ATPS component is dehydrated on a first portion of the LFA or in afirst layer of the flow-through assay (separation component). In someembodiments, the second phase solution component and/or second ATPScomponent is dehydrated on a second portion of the LFA or in a secondlayer of the flow-through assay (separation component). In someembodiments, the first portion and the second portion are same. In someembodiments, the first portion and the second portion are different. Byway of non-limiting example, in a PEG-salt ATPS, the PEG and saltsolutions can be dehydrated separately into different paper portions orsegments (see, e.g., FIG. 16 of copending PCT Application No:PCT/US2015/019297, filed on Mar. 6, 2015, which is hereby incorporatedby reference for the LFA configurations described therein) or inseparate layers comprising, e.g., the separation component of aflow-through assay (see, e.g., FIG. 4 ). In some embodiments,dehydrating the first/second phase solution and/or ATPS component ondifferent portions of the LFA or in different layers of the flow-throughassay provides a more uniform concentration of the first/second phasesolution components or ATPS components. In some embodiments, dehydratingthe first/second phase solution components and/or ATPS components ondifferent portions allows the first phase solution or ATPS component toflow in a first direction after hydration and the second phase solutionand/or ATPS component to flow in a second direction after hydration,wherein the first and second directions are different. In someembodiments, the target analyte is concentrated in the first direction,but not the second direction. In some embodiments, the target analyte isconcentrated in the second direction, but not the first direction. Insome embodiments, dehydrating the first/second phase components and/orATPS components on different portions allows the target analyte to flowin the first/second direction without requiring the sample to flow inthe first/second direction. In some embodiments, dehydrating thefirst/second phase components and/or ATPS components on differentportions allows the target analyte to flow faster, resulting indetection sooner. In some embodiments, dehydrating the first/secondphase components and/or ATPS components on different portions allows forincreased result reliability. In some embodiments, dehydrating thefirst/second phase components and/or ATPS components on differentportions prevents aggregation of first/second phase solution componentsand/or ATPS components (e.g. PEG-salt ATPS). In some embodiments, thefirst/second phase component and/or ATPS component is dehydrated inmultiple segments. In some embodiments the first/second phase componentand/or ATPS component is dehydrated in multiple segments, wherein thefirst/second phase component and/or ATPS component comprises a saltsolution. In some embodiments the first/second phase component and/orATPS component is dehydrated in multiple segments, wherein thefirst/second phase component and/or ATPS component does not comprise apolymer (e.g. PEG). In some embodiments, dehydrated PEG is not locatednear the detection zone because the PEG-rich phase can slow the flowwithin the detection membrane. In some embodiments, the LFA strip or theflow-through assay can comprise a blank spacer near the detection zonethat does not contain PEG or salt.

In some embodiments, a probe (e.g., an analyte binding moiety andassociated detection reagent/material) is provided in a probe buffer. Insome embodiments, the probe buffer is dehydrated on the LFA or in theflow-through assay.

In some embodiments, dehydration of ATPS components improves the limitof detection compared to a device in which the ATPS components are addedin liquid form. In some embodiments, the addition of liquid form ATPScomponents dilutes the sample solution from the subject. In someembodiments, dehydration of ATPS components allows for a distinct firstphase solution and/or distinct second phase solution to develop duringflow, concentrating the target analyte or probe-analyte complex in asmall volume at the front of the leading fluid that will reach the testand control lines or the detection component of a flow-through assay. Insome embodiments, concentrating the target analyte and or probe-analytecomplex at the front of the leading fluid will decrease the time periodnecessary for detection.

Probes

In certain embodiments the systems and/or devices described hereinand/or the methods described herein utilize a probe, where the probecomprises a binding moiety that binds the target analyte to form aprobe-analyte complex.

In some embodiments, the target analyte alone partitions preferentiallyinto the first phase solution or second phase solution or interface ofthe first phase solution and second phase solution. In some embodiments,the target analyte alone partitions extremely into the first phasesolution or second phase solution or interface of the first phasesolution and second phase solution.

In some embodiments, the target analyte alone does not partitionpreferentially into the first phase solution or second phase solution orinterface of the first phase solution and second phase solution. In someembodiments, the target analyte alone does not partition extremely intothe first phase solution or second phase solution or interface of thefirst phase solution and second phase solution.

In some embodiments, the probe-analyte complex partitions preferentiallyinto the first phase solution or second phase solution or interface ofthe first phase solution and second phase solution, thereby causing thetarget analyte (of the probe-analyte complex) to partitionpreferentially into the first phase solution or second phase solution orinterface of the first phase solution and second phase solution.

In some embodiments, the probe-analyte complex partitions extremely intothe first phase solution or second phase solution or interface of thefirst phase solution and second phase solution, thereby causing thetarget analyte (of the probe-analyte complex) to partition extremelyinto the first phase solution or second phase solution or interface ofthe first phase solution and second phase solution.

In some embodiments, the phrase “partitions preferentially,” when usedwith respect to the partitioning of the target analyte (or probe-analytecomplex) to a first/second phase solution of the ATPS, indicates that agreater amount of the target analyte becomes disposed in a preferredphase solution than in another phase solution of the ATPS.

In some embodiments, the phrase “partitions extremely,” when used withrespect to the partitioning of the target analyte (or probe-analytecomplex) to a first/second phase solution of the ATPS, indicates thatabout 90% or more of the target analyte becomes disposed in a preferredphase solution than in another phase solution of the ATPS.

In some embodiments, a greater amount of the target analyte partitionsinto the first phase solution. In some embodiments, greater than about50%, or greater than about 55%, or greater than about 60%, or greaterthan about 65%, or greater than about 70%, or greater than about 75%, orgreater than about 80%, or greater than about 85%, or greater than about90%, or greater than about 95%, or greater than about 98%, or greaterthan about 99% of the target analyte partitions into the first phasesolution. In some embodiments, greater than about 99%, or greater thanabout 99.1%, or greater than about 99.2%, or greater than about 99.3%,or greater than about 99.4%, or greater than about 99.5%, or greaterthan about 99.6%, or greater than about 99.7%, or greater than about99.8%, or greater than about 99.9% of the target analyte partitions intothe first phase solution.

In some embodiments, a greater amount of the analyte partitions into thesecond phase solution. In some embodiments, greater than about 50%, orgreater than about 55%, or greater than about 60%, or greater than about65%, or greater than about 70%, or greater than about 75%, or greaterthan about 80%, or greater than about 85%, or greater than about 90%, orgreater than about 95%, or greater than about 98%, or greater than about99% of the target analyte partitions into the second phase solution. Insome embodiments, greater than about 99%, or greater than about 99.1%,or greater than about 99.2%, or greater than about 99.3%, or greaterthan about 99.4%, or greater than about 99.5%, or greater than about99.6%, or greater than about 99.7%, or greater than about 99.8%, orgreater than about 99.9% of the target analyte partitions into thesecond phase solution.

In some embodiments, a greater amount of the analyte partitions into theinterface of the first phase solution and the second phase solution. Insome embodiments, greater than about 50%, or greater than about 55%, orgreater than about 60%, or greater than about 65%, or greater than about70%, or greater than about 75%, or greater than about 80%, or greaterthan about 85%, or greater than about 90%, or greater than about 95%, orgreater than about 98%, or greater than about 99% of the target analytepartitions into the interface. In some embodiments, greater than about99%, or greater than about 99.1%, or greater than about 99.2%, orgreater than about 99.3%, or greater than about 99.4%, or greater thanabout 99.5%, or greater than about 99.6%, or greater than about 99.7%,or greater than about 99.8%, or greater than about 99.9% of the targetanalyte partitions into the interface.

In some embodiments, the device comprises or is configured to utilizeand/or the assay run on the device utilizes 1 probe. In someembodiments, the device comprises or is configured to utilize and/or theassay run on the device utilizes at least two different probes, or atleast 3 different probes, or at least 4 different probes, or at least 5different probes, or at least 7 different probes, or at least 10different probes, or at least 15 different probes, or at least 20different probes.

In some embodiments, the probe comprises one or more of a syntheticpolymer, a metal, a mineral, a glass, a quartz, a ceramic, a biologicalpolymer, a plastic, and/or combinations thereof. In some embodiments,the probe comprises a polymer comprises a polyethylene, polypropylene,nylon (DELRIN®), polytetrafluoroethylene (TEFLON®), dextran andpolyvinyl chloride. In some embodiments, the polyethylene ispolyethylene glycol. In some embodiments, the polypropylene ispolypropylene glycol. In some embodiments, the probe comprises abiological polymer that comprises one or more of a collagen, cellulose,and/or chitin. In some embodiments, the probe comprises a metal thatcomprises one or more of gold, silver, platinum titanium, stainlesssteel, aluminum, or alloys thereof. In some embodiments, the probecomprises a nanoparticle (e.g., a gold nanoparticle, a silvernanoparticle, etc.).

In some embodiments, the probe further comprises a coating. In someembodiments, the coating comprises polyethylene glycol or polypropyleneglycol. In some embodiments, the coating comprises polypropylene. Insome embodiments, the coating comprises polypropylene glycol. In someembodiments, the coating comprises dextran. In some embodiments, thecoating comprises a hydrophilic protein. In some embodiments, thecoating comprises serum albumin. In some embodiments, the coating has anaffinity for the first phase solution or the second phase solution.

In some embodiments, the amount of target analyte in the sample is verylow, such that the analyte needs to be substantially concentrated toenable detection by LFA or flow-through assay. In certain embodiments,substantial concentration is achieved at an interface, since the degreeof analyte concentration is dependent on the volume of a phase in whichthe analyte partitions, or concentrates, and the “volume” at theinterface is very small relative to the bulk phases.

In some embodiments, the probe partitions preferentially (or extremely)to the interface in order to drive the target analyte towards aninterface. In some embodiments, the probe partitions preferentially (orextremely) to the interface due to their surface chemistry, wherein thesurface chemistry is optimized to drive the probe to the interface. Byway of non-limiting example, to drive the probe-analyte complex to theinterface of a polymer-salt ATPS system, such as the polyethyleneglycol-potassium phosphate (PEG/salt) system, the probes are conjugatedto PEG (or PEGylated) to promote the PEG-PEG interaction with thePEG-rich phase, and/or are decorated with hydrophilic proteins topromote hydrophilic interactions with the PEG-poor phase. Using such anoptimized probe decorated with specific antibodies or other moleculescapable of binding to the target, the target analyte is captured andcollected at the interface. Since the volume of the interface is verysmall, the analytes are highly concentrated and are applied to thesubsequent LFA or detection region of the flow-through assay.

In some embodiments, gold nanoprobes (GNP) are prepared that are capableof partitioning to the interface of a PEG/salt ATPS, and operatingconditions are optimized to allow for a fast phase separation time witha very high recovery of GNP/analyte.

In some embodiments, the probe-analyte complex partitions to asolid-liquid interface in the ATPS. In some embodiments, the solid isthe wall of the chamber that contains the ATPS. In some embodiments, thesolid is the collector of the assay device. In some embodiments, thesolid comprises a solid polymer. In some embodiments, the solid polymercomprises polyethylene, cellulose, chitin, nylon, polyoxymethylene(DELRIN®), polytetrafluoroethylene (TEFLON®), polyvinyl chloride, orcombinations thereof. In some embodiments, the solid polymer comprisespolypropylene. In some embodiments, the probe-analyte complex sticks tothe solid and is highly concentrated since it is present in the smallvolume at the solid-liquid interface, and not diluted by the volume ofthe bulk phases. In some embodiments, the bulk phase is removed withoutdisrupting the concentrated analyte, and is collected by washing, withsubsequent application to the LFA or to the flow-through assay device.In some embodiments, this approach significantly concentrates theanalyte and allows collection without using an external force (e.g.,magnet). Alternatively, the probe comprises a magnetic material and thisapproach is used with a magnet. In some embodiments, these probes aremodified to be concentrated at the interface for extreme analyteconcentration. As mentioned above, this approach can provide additionalseparation of the target analyte from other contaminants, which isnonspecifically concentrated by ATPS, through the use of a magnet. Insome embodiments, the ATPS concentration enables the magnetic probe towork more efficiently, since the magnetic probe would first beconcentrated into a very small volume at a specific location (theinterface). Accordingly, a smaller magnet or a weaker magnetic fieldwill be required to collect the concentrated analyte. In someembodiments, the combination of ATPS interface concentration withmagnetic probes allows for the development of a more effective, rapid,and cheaper device compared to the current state-of-the-art.

Binding Moiety

In some embodiments, the binding moiety is a molecule that binds thetarget analyte (e.g., bacterium, fungus, virus, etc.). In someembodiments, the binding moiety is a molecule that specifically bindsthe target analyte. In some embodiments, “specifically binds” indicatesthat the molecule binds preferentially to the target analyte or bindswith greater affinity to the target analyte than to other molecules. Byway of non-limiting example, an antibody will selectively bind to anantigen against which it was raised. Also, by way of non-limitingexample, a DNA molecule will bind to a substantially complementarysequence and not to unrelated sequences under stringent conditions. Insome embodiments, “specific binding” can refer to a binding reactionthat is determinative of the presence of a target analyte in aheterogeneous population of molecules (e.g., proteins and otherbiologics). In some embodiments, the binding moiety binds to itsparticular target analyte and does not bind in a significant amount toother molecules present in the sample.

In some embodiments, the binding moiety comprises an antibody, a lectin,a protein, a glycoprotein, a nucleic acid, monomeric nucleic acid, apolymeric nucleic acid, an aptamer, an aptazyme, a small molecule, apolymer, a lectin, a carbohydrate, a polysaccharide, a sugar, a lipid,or any combination thereof. In some embodiments, the binding moiety is amolecule capable of forming a binding pair with the target analyte.

In some embodiments, the binding moiety is an antibody or antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, Fv′, Fd, Fd′, scFv, hsFv fragments, cameloidantibodies, diabodies, and other fragments described below.

In some embodiments, an “antibody” refers to a protein consisting of oneor more polypeptides substantially encoded by immunoglobulin genes orfragments of an immunoglobulin gene. As used herein, the terms“antibody” and “immunoglobulin” are used interchangeably, unlessotherwise specified. In some embodiments, the immunoglobulin gene is animmunoglobulin constant region gene. In some embodiments, theimmunoglobulin gene, is by non-limiting example, a kappa, lambda, alpha,gamma, delta, epsilon or mu constant region gene. In some embodiments,the immunoglobulin gene is an immunoglobulin variable region gene. Insome embodiments, the immunoglobulin gene comprises a light chain. Insome embodiments, the light chain comprises a kappa light chain, alambda light chain or a combination thereof. In some embodiments, theimmunoglobulin gene comprises a heavy chain. In some embodiments, theheavy chain is classified as gamma, mu, alpha, delta, or epsilon, whichin turn correspond to the immunoglobulin classes, IgG, IgM, IgA, IgD andIgE, respectively.

In some embodiments, the immunoglobulin comprises a tetramer. In someembodiments, the tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). In some embodiments, the N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (V_(L)) and variable heavy chain (V_(H)) refer to theselight and heavy chains, respectively.

In some embodiments, the antibody comprises an intact immunoglobulin. Insome embodiments, the antibody comprises a number of well characterizedfragments produced by digestion with various peptidases. In someembodiments, the peptidase is pepsin. In some embodiments, the pepsindigests a disulfide linkage in the hinge region to produce F(ab)′₂, adimer of Fab which itself is a light chain joined to V_(H)-C_(H)1 by adisulfide bond. In some embodiments, the F(ab)′₂ is reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)₂ dimer into a Fab′ monomer. In some embodiments,the Fab′ monomer consists essentially of a Fab with part of the hingeregion. In some embodiments, the Fab′ fragment is synthesized de novoeither chemically or by utilizing recombinant DNA methodology. In someembodiments, the antibody fragment is produced by the modification of awhole antibody. In some embodiments, the antibody fragment issynthesized de novo using recombinant DNA methodologies. In someembodiments, the antibody includes a single chain antibody (antibodiesthat exist as a single polypeptide chain). In some embodiments, theantibody includes a single chain Fv antibody (sFv or scFv) in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide. In someembodiments, the antibody includes a single chain Fv antibody. In someembodiments, the antibody comprises a covalently linked V_(H)-V_(L)heterodimer which may be expressed from a nucleic acid including V_(H)-and V_(L)-encoding sequences either joined directly or joined by apeptide-encoding linker. In some embodiments, the V_(H) and V_(L) areconnected to each as a single polypeptide chain, and the V_(H) and V_(L)domains associate non-covalently. In some embodiments, the Fab isdisplayed on a phage, wherein one of the chains (heavy or light) isfused to g3 capsid protein and the complementary chain exported to theperiplasm as a soluble molecule. In some embodiments, the two chains canbe encoded on the same or on different replicons. In some embodiments,the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p. In someembodiments, the antibody has been displayed on a phage or yeast.

In some embodiments, the antibody fragment is derived via proteolyticdigestion of intact antibodies. In some embodiments, the antibodyfragment is produced directly by recombinant host cells. In someembodiments, the Fab, Fv or scFv antibody fragment is expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of these. In some embodiments, the antibody fragment is isolatedfrom antibody phage libraries. In some embodiments, the Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments. In some embodiments, the F(ab′)₂ fragment isisolated directly from recombinant host cell culture. In someembodiments, the Fab and F(ab′)₂ fragments have an increased in vivohalf-life. In some embodiments, the Fab and F(ab′)₂ fragments comprisesalvage receptor binding epitope residues. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, the antibody of choice is a singlechain Fv fragment. In some embodiments, the Fv or sFv has an intactcombining site that is devoid of a constant region; thus, it is suitablefor reduced non-specific binding during in vivo use. In someembodiments, the antibody fragment is a “linear antibody.” In someembodiments, the linear antibody fragment is monospecific. In someembodiments, the linear antibody fragment is bispecific.

In some embodiments, the antibody fragment is a diabody. In someembodiments, the diabody is an antibody fragment with two antigenbinding sites that may be bivalent or bispecific.

In some embodiments, the antibody fragment is a single-domain antibody.In some embodiments, the single-domain antibody is an antibody fragmentcomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody.

In certain embodiments, the binding moiety comprises an aptamer. In someembodiments, the aptamer comprises an antibody-analogue formed fromnucleic acids. In some embodiments, the aptamer does not require bindingof a label to be detected in some assays, such as nano-CHEM-FET, wherethe reconfiguration would be detected directly. In some embodiments, thebinding moiety comprises an aptazyme. In some embodiments, the aptazymecomprises an enzyme analogue, formed from nucleic acids. In someembodiments, the aptazyme functions to change configuration to capture aspecific molecule, only in the presence of a second, specific, analyte.

In some embodiments, the probe comprises a detectable label. Detectablelabels include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical, orchemical means. Illustrative useful labels include, but are not limitedto, fluorescent nanoparticles (e.g., quantum dots (Qdots)), metalnanoparticles, including but not limited to gold nanoparticles, silvernanoparticles, platinum nanoparticles, fluorescent dyes (e.g.,fluorescein, texas red, rhodamine, green fluorescent protein, and thelike, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In,^(113m)In, ⁹⁷Ru, ⁶²Cu, 64lCu, ⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As,⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb,¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag, and the like), enzymes (e.g., horse radishperoxidase, alkaline phosphatase and others commonly used in an ELISA),various colorimetric labels, magnetic or paramagnetic labels (e.g.,magnetic and/or paramagnetic nanoparticles), spin labels, radio-opaquelabels, and the like.

Alternatively or additionally, the probe can bind to another particlethat comprises a detectable label. In some embodiments, the probesprovide a detectable signal at the detection zone (e.g., test line,control line, test region, control region). In some embodiments, thedetectable label/property comprises one or more of a colorimetriclabel/property, a fluorescent label/property, an enzymaticlabel/property, a colorigenic label/property, and/or a radioactivelabel/property. In some embodiments, the probe is a gold nanoparticleand the detectable property is a color. In some embodiments, the coloris orange, red or purple.

Sample Collection

In various embodiments the sample to be assayed using the devices andmethods described herein comprises a biological sample. Illustrativebiological samples include, but are not limited to biofluids such asblood or blood fractions, lymph, cerebrospinal fluid, seminal fluid,urine, oral fluid, vaginal fluid, and the like, tissue samples, plaquesamples, vaginal swab samples, endocervical swab samples, cell samples,tissue or organ biopsies or aspirates, histological specimens, and thelike.

Where the biological sample comprises a tissue, in certain embodiments,the tissue may be lysed, homogenized, and/or ground and, optionallysuspended in a sample solution. Where the biological sample comprise abiological fluid the fluid may be assayed directly or suspended in asample solution prior to assay. In certain embodiments the samplesolution may act to preserve or stabilize the biological sample orcomponents thereof, and/or may act to extract or concentrate thebiological sample or components thereof. In certain embodiments thesample solution may comprise a buffer, optionally containingpreservatives, and/or enzymes (protease, nuclease, etc.), and/orsurfactants, and/or ATPS components.

In certain embodiments, particular in point-of-care embodiments, thesample may be applied to the assay device immediately or after a modesttime interval. In certain embodiments the sample may be delivered to aremote testing facility where the assay is run.

Methods and devices for collecting biological samples are well known tothose of skill in the art, e.g., as illustrated below:

Oral Fluid Collection

Oral fluid can be collected by drooling into an empty vial, thentransferring the fluid to the concentration component of the assay.

Oral fluid can also be collected using a swab and/or collection pad. Forexample, a swab or a collection pad can be placed in the user's mouth tosoak up the oral fluid. The swab or the collection pad may containcompounds, such as peppermint extract, or a sour extract, to stimulateoral fluid production. The swab or collection pad can also act as afilter to remove food debris, contaminants, or mucus that may affect thedownstream concentration and detection steps. In certain embodiments theoral fluid in the swab or collection pad can be extracted and mixed withaqueous two-phase components (ATPS) components for concentration.Extraction of the oral fluid from the collection device can beaccomplished, for example, by applying physical pressure to the swab/padto squeeze the fluid out, or by capillary action to introduce the fluidto the concentration component. Another configuration corresponds to theATPS components being dehydrated downstream of the swab or collectionpad so that no further user interaction is necessary.

Plaque Collection

Plaque can be collected by brushes, swabs, or picks on the surfaces ofteeth, underneath gum, or between teeth. In certain embodiments thecollected plaque can then be mixed in buffer or an ATPS solution forsubsequent concentration.

Urine Collection

In various embodiments urine can be obtained with a collection cup. Thecollected urine can then be mixed in an ATPS solution for subsequentconcentration, or applied directly onto the device if ATPS componentsare dehydrated in the concentration component. In a catheterizedsubject, urine can be obtained from the catheter or from the catheterreceiving bag.

Vaginal/Endocervical Swab

Target analytes on the vaginal or cervical surface and/or in vaginalfluid can be collected by commercially available swabs. The collectedswab can be placed in a buffer to release the target, or placed in theATPS solution for direct concentration of the target biomolecules.

Blood Collection

Blood can be collected by pin (lancet) prick and collection in acapillary tube, by syringe, and the like.

Illustrative Analytes.

While essentially any analyte can be detected and/or quantified usingthe assay devices and methods described herein, in certain embodiments,the analyte is a clinically relevant analyte (e.g., a bacterium, afungus, a protozoan, an amoeba, a virus, and the like).

Clinically relevant targets are well known to those of skill in the art.

Clinically Important Bacteria in Vaginal Fluids

Finding Trichomonas vaginalis, bacterial vaginosis and Actinomycesinfections in vaginal fluid or tissue samples, pap smears might beconsidered an indication for treatment without performing otherdiagnostic tests. Treatment of asymptomatic infections can preventcomplications in selected patients. Candida can be a commensal bacteriain the vagina, therefore asymptomatic patients may not requiretreatment. Detection of a higher rate of Trichomonas vaginalis andcandida infection in intrauterine device (IUD) users shows that IUDs canincrease the risk of vaginal infections and associated complications.

Gonorrhea is a bacterial infection caused by the organism Neisseriagonorrheae and is a clinically important pathogen. Similarly, Chlamydia,caused by Chlamydia trachomatis and syphilis, caused by Treponemapallidum are important sexually transmitted disease whose rapiddiagnosis is desirable.

Clinically Important Bacteria in Urine

Escherichia coli and Proteus sp. are bacterial pathogens that when foundin urine are typically indicative of urinary tract infections.

Clinically Important Bacteria in the Oral Cavity

Gram-negative oral anaerobes have frequently been associated withperiodontal disease, some species more frequently than others. Suchanerobes include, but are not limited to Prevotella species (e.g., Pr.intermedia, Pr. nigrescens, Pr. melaninogenica, Pr. veroralis, and thelike) and Porphyromonas species (e.g., Porph. gingivalis).

Additionally Streptococcus mutans has been implicated in the formationof dental caries. Additional clinically important bacteria of theinstant disclosure include but are not limited to Actinomyces viscosus,Lactobacillus casei, Staphylococcus aureus, Candida albicans,Lactobacillus acidophilus, Capnocytophaga gingivalis, Fusobacteriunnicleatum, or Bacteroides fortsythus.

It will be recognized that these pathogens are illustrative andnon-limiting. One of skill will recognize that the assay devices andmethods described herein can be used to detect and/or to quantifynumerous other analytes.

Kits.

In certain embodiments kits are provided for use of the devices and/orpractice of the methods described herein. In certain embodiments a kitfor the detection and/or quantification of an analyte is provided wherethe kit comprises a container containing an assay device as describedherein. In certain embodiments the kit additionally contains acollection device for collecting a sample. In certain embodiments thecollection device comprises a device for collecting oral fluid, a devicefor collecting blood, a urine collection device, a device for collectingvaginal fluid or from a vaginal swab or from an endocervical swab, or adevice for collecting an environmental sample.

In certain embodiments the kits additionally contain reagents such asbuffers, solvents, components of an ATPS system, detection reagents, andthe like.

In certain embodiments the kits additionally contain instructionalmaterials providing methods (e.g., protocols) for use of the assaydevices provided therein. Often and typically the instructionalmaterials are provided in written form and can be printed on the kitcomponents themselves (e.g. on the cover of a box, container, or on anenvelope, or can be provided as an insert/instructional page or booklet.While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplated.Such media include, but are not limited to electronic storage media(e.g., magnetic discs, tapes, cartridges, chips, flash memory), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Detection of Streptococcus mutans

Goal

We incorporated ATPS and LFA into a single paper-based diagnostic deviceused to detect Streptococcus mutans (S. mutans), which is the dominantbacterium that could lead to dental caries (cavities). Previously, wewere able to use a micellar ATPS to achieve 10-fold concentration of S.mutans and improve the detection limit of LFA by 10-fold. In thesestudies, we examined other systems for this process. Specifically, weinvestigated the PPG/salt ATPS, which phase separates more quickly thanthe micellar ATPS and the polyethylene glycol (PEG)/salt ATPS in testtube solutions. The PPG/salt ATPS also requires less salt to achievephase separation which provides an even more suitable environment forbiomolecules to bind to the probes and for the probes to bind at thetest line.

Methods and Materials

Preparing the Anti-S. mutans DGNPs

The pH of a 1 mL dextran-coated gold nanoparticle (DGNP) solution wasfirst adjusted to pH 9 using 1.5 N NaOH. Subsequently, 16 μg of mousemonoclonal S. mutans antibody were added to the gold solution and mixedfor 30 min on a shaker. To prevent nonspecific binding of other proteinsto the surfaces of the colloidal gold nanoparticles, 200 μL of a 10% w/vbovine serum albumin (BSA) solution were added to the mixture and mixedfor 20 min on a shaker. To remove free, unbound antibodies, the mixturewas then centrifuged for 30 min at 4° C. and 9,000 rpm, followed byresuspending the pellet of DGNPs in 200 μL of a 1% w/v BSA solution. Thecentrifugation and resuspension steps were repeated two more times, andafter the third centrifugation, the pellet of DGNPs was resuspended in100 μL of 0.1 M sodium borate buffer at pH 9.0.

Detection Using LFA

LFA test strips utilizing the sandwich assay format were assembled in asimilar manner to our previous studies described in copending PCTApplication No: PCT/US2015/019297, filed on Mar. 6, 2015, which isincorporated herein by reference for the assay formats and reagentsdescribed herein. In this format, immobilized S. mutans antibodyconstituted the test line and immobilized secondary antibodies specificto the primary antibody constituted the control line.

To verify the detection limit of S. mutans with LFA, DGNPs were added toa sample solution and allowed to bind S. mutans present in the sample. Asample suspension containing some saliva, DGNPs, and knownconcentrations of S. mutans were mixed in a test tube. The LFA teststrip was inserted vertically into each sample suspension, which wickedupward through the strip via capillary action towards the absorbent pad.Images of the test strips were taken after 10 min in a controlledlighting environment.

Detection Using LFA with ATPS

A PPG/potassium phosphate ATPS sample solution with a top phase tobottom phase volume ratio of 60:1 was prepared, which consisted of knownconcentrations of S. mutans. The ATPS sample solutions were incubated atroom temperature for 10 min to allow phase separation to occur. Thebottom PEG-poor phase which contained concentrated S. mutans wasextracted and incubated with anti-S. mutans DGNP. The LFA test strip wasinserted vertically into the resulting mixture, and images of the teststrips were taken after 10 min in a controlled lighting environment(FIG. 5 ).

We successfully concentrated S. mutans using the new ATPS anddrastically improved the concentration factor from 10-fold to 60-fold.The phase separation time also improved from hours (in a test tubesolution containing a micellar ATPS) to only 10 min (in a test tubesolution containing the PPG/salt ATPS). We then demonstrated thisenhancement can be applied to the subsequent detection step and showed a60-fold improvement in detection limit of LFA (FIG. 5 ), reaching 1×10⁵cells/mL. The entire assay was completed within 20 min.

Example 2 Detection of Streptococcus mutans in Plaque

Goal

We investigated the feasibility of detecting S. mutans in plaque.

Methods and Materials

Toothpicks were used to extract plaque from subjects. The collectedplaque was then dissolved in phosphate-buffered saline (PBS). LFA teststrip, prepared as described above, was applied to the resultingsolution. Images of the test strips were taken after 10 min in acontrolled lighting environment.

Results.

FIG. 6 shows the detection of S. mutans in plaque from 4 subjects. Thehigher test line intensity indicates a greater concentration of S.mutans in the subject.

FIG. 7 shows the detection of S. mutans in plaque before and afterbrushing teeth. The results indicated that brushing teeth is effectivein removing S. mutans and lowering the risk to develop dental caries.

Example 3 Chlamydia Detection

Goal

We incorporated ATPS and LFA into a single paper-based diagnostic devicethat could be used to detect Chlamydia trachomatis (C. trachomatis) in apatient urine or a patient swab sample.

Methods and Materials

Preparing the Anti-C. trachomatis DGNPs

The pH of a 1 mL dextran-coated gold nanoparticle (DGNP) solution wasfirst adjusted to pH 9 using 1.5 N NaOH. Subsequently, 16 μg of mousemonoclonal C. trachomatis antibody were added to the colloidal goldsuspension and mixed for 30 min on a shaker. To prevent nonspecificbinding of other proteins to the surfaces of the colloidal goldnanoparticles, 200 μL of a 10% w/v bovine serum albumin (BSA) solutionwere added to the mixture and mixed for 20 min on a shaker. To removefree, unbound antibodies, the mixture was then centrifuged for 30 min at4° C. and 9,000 rpm, followed by resuspending the pellet of DGNPs in 200μL of a 1% w/v BSA solution. The centrifugation and resuspension stepswere repeated two more times, and after the third centrifugation, thepellet of DGNPs was resuspended in 100 μL of 0.1 M sodium borate bufferat pH 9.0.

Detection Using LFA

LFA test strips utilizing the sandwich assay format were assembled in asimilar manner to our previous studies. In this format, immobilized C.trachomatis antibody constituted the test line and immobilized secondaryantibodies specific to the primary C. trachomatis antibody constitutedthe control line.

To verify the detection limit of C. trachomatis with LFA, DGNPs wereadded to a sample solution and allowed to bind C. trachomatis present inthe sample. A suspension containing DGNPs in phosphate-buffered saline(PBS) and a solution containing a known concentration of C. trachomatisin PBS were mixed in a test tube. The LFA test strip was insertedvertically into the sample solution, which wicked through the strip viacapillary action upward towards the absorbance pad. Images of the teststrips were taken after 10 min in a controlled lighting environment.

Detection Using LFA with ATPS

A PEG/potassium phosphate ATPS sample solution with a top phase tobottom phase volume ratio of 9:1 was prepared, which consisted of knownconcentrations of C. trachomatis. The ATPS sample solutions wereincubated at room temperature for 30 min to allow phase separation tooccur. The bottom PEG-poor phase which contained concentrated C.trachomatis was extracted and incubated with anti-C. trachomatis DGNP.The LFA test strip was inserted vertically into the resulting mixture.Images of the test strips were taken after 10 min in a controlledlighting environment.

Results

FIG. 8 shows detection of C. trachomatis in PBS using LFA alone andusing ATPS with LFA. FIG. 9 shows the performance of our device comparedwith an FDA approved, commercially available chlamydia LFA for aclinical urine sample collected from a C. trachomatis positive patient.Our device is able to provide a true positive result (the presence ofthe test line), while the commercial test gave a false negative result(the absence of the test line).

Example 4 An Illustrative Diagnostic Device with Dehydrated ATPSComponents

In one illustrative embodiment, a dehydrated ATPS diagnostic device(FIG. 11 ) is comprised of two major components: The ATPS rehydrationand resolubilization optimized wick (ARROW) and the standard LFA. In theillustrated embodiment, the ARROW consists of 5 fiberglass paper sheetslayered together. Considering that the function of the ATPS is toconcentrate the target pathogen, it was important that the ARROW wasable to wick up a large volume of sample solution. Each sheet is firstpre-treated with BSA in order to prevent non-specific binding of C.trachomatis during sample solution flow. After pre-treatment, 20 μL of15% (w/w) potassium phosphate was dehydrated in the upstream portion ofeach fiberglass sheet, while 30 μL of 10% (w/w) PEG 8000 was dehydratedin the downstream portion of each fiberglass sheet. It is important toleave blank space between the dehydrated PEG and the tip of the sheet toallow for PEG-poor phase collection, which contains the concentratedpathogen. The downstream tip of each sheet is tapered to form a point,which facilitates proper transition of the liquid into the conjugatepad.

The LFA portion of the diagnostic consisted of the conjugate padcontaining the colorimetric indicator, connected to a nitrocellulosemembrane with printed primary and secondary antibodies, and followed byan absorbance pad. The LFA portion interfaced with the ARROW by fittinga small upstream portion of the conjugate pad perpendicularly into aslit that had been cut in the ARROW.

SEM images (FIG. 11 ) of the blank fiberglass region of the fiberglasspaper shows a porous fiber-based matrix structure. The dehydrated PEGand potassium phosphate regions show a similar porous structure, withthe addition of web-like connections, which it is believed contains amajority of their respective ATPS components. These images demonstratethat the process of dehydration does not significantly deform the porousstructure of the fiberglass paper, which is critical for proper wickingof the sample fluid.

Importance of the Rehydration Order of PEG and Potassium Phosphate

The effect of the PEG and potassium phosphate rehydration order on thephase separation behavior within the paper was investigated. To do this,a suspension comprised of BSA-DGNPs and Brilliant Blue dye was utilizedwhich allowed visualization of the phase separation process as thesuspension flowed through the paper. In short, the BSA-DGNPs partitioninto the PEG-poor phase indicated by the burgundy/light purple color,while the blue dye partitions into the PEG-rich phase indicated by thelight blue color. Regions of macroscopically mixed domains containedboth BSA-DGNPs and blue dye, indicated by the dark blue/dark purplecolor. During fiberglass paper preparation, the location of thedehydrated ATPS components was altered, such that one condition had thedehydrated potassium phosphate located upstream of the dehydrated PEG(denoted “Salt→PEG”), while the other condition had the dehydrated PEGlocated upstream of the dehydrated potassium phosphate (denoted“PEG→Salt”).

From these results (FIG. 12 ), we note two interesting observations.First, the leading PEG-poor fluid had a significantly darker burgundycolor in the ‘Salt→PEG’ condition compared to the ‘PEG→Salt’ condition,indicating that the ‘Salt→PEG’ condition contained more BSA-DGNPs in theleading fluid, and therefore, is more effective at concentrating largespecies. Second, the PEG-rich phase, the area identified by the dashedlines in FIG. 12 , exhibited significantly more volumetric growth overtime in the ‘Salt→PEG’ condition compared to the PEG-rich phase in the‘PEG→Salt’ condition. This suggests that in the ‘Salt→PEG’ condition,the newly formed PEG-poor domains are able to get out of the mixeddomain region and more efficiently pass through the trailing PEG-richphase and collect into the leading PEG-poor phase. is results in thePEG-rich phase becoming larger as the mixed domains region becomessmaller. One possible reason for this phenomenon is the formation ofPEG-poor channels within the PEG-rich phase that connect to the leadingPEG-poor phase. Research in multiphase fluid flow within porous mediahas found that less viscous fluids will develop preferred channels whendisplacing more viscous fluids.

Improved Limit of Detection for C. trachomatis Using the Integrated LFAand ARROW

We demonstrated that our ARROW design effectively concentrated a C.trachomatis sample suspension, resulting in an improved limit ofdetection for LFA. To do this, we ran sample solutions of varyinginitial concentrations of C. trachomatis on LFA test strips, with andwithout the ARROW component. We see from the results of the LFA panel(FIG. 13 ) that the LFA only system started showing false negativeresults at around 15.8 ng/μL C. trachomatis while the integrated LFA andARROW system started showing false negative results at around 1.58 ng/μLC. trachomatis. This visually demonstrates a 10-fold improvement in thelimit of detection.

We also quantified the pixel contrast of the test lines on the LFAimages using a customized MATLAB program (FIG. 14 ). This allowed us toquantitatively assess the improvement in the limit of detection. For anygiven concentration of C. trachomatis, we see a significant increase inthe test line intensity for the integrated ARROW and LFA system comparedto the LFA only system. For example, at 50 ng/μL C. trachomatis, the LFAonly condition had a pixel contrast intensity of 8.3±1.7, while theintegrated ARROW and LFA had a pixel contrast intensity of 37.6±0.6.Furthermore, we also see confirmation of our panel results where thesame test line intensity 8.3 was observed at the limits of detectionnoted in the panels (50 ng/μL for LFA alone and 5 ng/μL for integratedARROW and LFA).

We wanted to verify that these quantities were in fact physiologicallyrelevant. Using remnant clinical urine specimens, our urine-based LFA(by itself) had poor sensitivity, consistent with what we obtained withthe QuickVue test for samples in urine. However, when the urine-basedLFA was integrated with ATPS, we demonstrated a significant improvementin sensitivity, recognizing 87.5% (14/16) CT+ urine samples with apositive result. (Table 2). An example of this head-to-head comparisonis shown in FIG. 15 .

TABLE 2 A summary of a performance comparison study between FDA approvedQuickVue, phase diagnostics' LFA-only test, and phase diagnostics' LFA +ATPS test in the detection of ct in remnant clinical urine samples. Allsamples were ct+ confirmed by a nucleic acid amplification test (NAAT).In contrast to the frozen samples, neat samples were freshly collectedones. Phase's Sample Type NAAT QuickVue Phase's LFA LFA + ATPS 1 Neat +− + + 2 Neat + − − + 3 Neat + − − + 4 Neat + − − + 5 Neat + − − + 6Neat + − − + 7 Frozen + − − + 8 Frozen + − − + 9 Frozen + − − + 10Frozen + − − + 11 Frozen + − − − 12 Frozen + − − − 13 Frozen + − − + 14Frozen + − − + 15 Frozen + − − + 16 Frozen + − − +

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of concentrating an analyte using anaqueous two-phase system, said method comprising: providing a porousmatrix comprising salt-polymer aqueous two-phase system (ATPS)comprising a mixed phase solution that separates into a first phasesolution and a second phase solution as said ATPS moves through saidporous matrix wherein said ATPS is provided as a dehydrated salt and adehydrated polymer that when rehydrated form said mixed phase of anaqueous two-phase system, where said dehydrated salt is disposed in saidporous matrix upstream from said dehydrated polymer; applying a samplesolution comprising said analyte to said porous matrix where said samplesolution rehydrates the dehydrated salt and dehydrated polymer to formthe mixed phase solution that separates into a first phase solution anda second phase solution as said ATPS moves through said porous matrixand concentrates the analyte into the first phase solution or the secondphase solution or an interface between the first phase solution andsecond phase solution.
 2. The method of claim 1, wherein said porousmatrix comprises a material selected from the group consisting offiberglass, cellulose, and nitrocellulose.
 3. The method of claim 2,wherein said porous matrix comprises fiberglass.
 4. The method of claim1, wherein said polymer comprises a polymer selected from the groupconsisting of polyethylene glycol (PEG), polypropylene glycol, anddextran.
 5. The method of claim 4, wherein said polymer comprisespolyethylene glycol.
 6. The method of claim 1, wherein said saltcomprises a salt selected from the group consisting of potassiumphosphate, sodium sulfate, magnesium sulfate, ammonium sulfate, andsodium citrate.
 7. The method of claim 6, wherein said salt comprisespotassium phosphate.
 8. The method of claim 1, wherein: said polymercomprise polyethylene glycol (PEG); and said salt comprises potassiumphosphate.
 9. The method of claim 8, wherein said porous matrixcomprises a material selected from the group consisting of fiberglass,cellulose, and nitrocellulose.
 10. The method of claim 9, wherein saidporous matrix comprises fiberglass.
 11. The method of claim 1, whereinsaid analyte comprises an analyte selected from the group consisting ofa bacterium, a fungus, and a virus.
 12. The method of claim 11, whereinsaid analyte comprises a bacterium.
 13. The method of claim 12, whereinsaid analyte comprises an oral bacterium, a bacterium found in urine, abacterium found in vaginal fluid, or a bacterium found on a vaginalswab, or a bacterium found on an endocervical swab.
 14. The method ofclaim 13, wherein said analyte comprises an oral bacterium.
 15. Themethod of claim 14, wherein said analyte comprises an oral bacteriumselected from the group consisting of Prevotella sp., Porphyromonas sp.,Streptococcus sp., Actinomyces viscosus, Lactobacillus casei,Staphylococcus aureus, Candida albicans, Lactobacillus acidophilus,Capnocytophaga gingivalis, Fusobacterium nucleatum, and Bacteroidesfortsythus.
 16. The method of claim 13, wherein said analyte comprises abacterium found in vaginal fluid.
 17. The method of claim 16, whereinsaid analyte comprises a bacterium selected from the group consisting ofTrichomonas sp., Actinomyces sp., Gardnerella sp., Neisseria sp.,Chlamydia sp., or Treponema sp.
 18. The method of claim 13, wherein saidanalyte comprises a bacterium found in urine.
 19. The method of claim18, wherein said analyte comprises a bacterium selected from the groupconsisting of E. coli, Proteus sp., Trichomonas sp., Actinomyces sp.,Gardnerella sp., Neisseria sp., Chlamydia sp., or Treponema sp.